CN1131016C - Simple and efficient freeze-thaw method for vitrification of embryo - Google Patents

本发明公开了一种简易高效胚胎玻璃化冷冻解冻方法，属胚胎生物技术领域。本方法速度快、成本低、操作简单、效率高。本发明采用乙二醇(EG)或丙三醇(GL)为主体抗冻保护剂配制成EFS或GFS玻璃化溶液，浓度大于7.0Mol/L，在室温(20～25℃)下，将胚胎于10～20%EG(或GL)溶液预处理5～10min，然后移入EFS(或GFS)液中平衡30s～1min后直接投入液氮。本方法冷冻保存的小鼠原核胚～孵化囊胚，其成活率均能达到95%左右，并获得了较高的移植妊娠率(60～75%)。本方法对其他家畜如兔、牛和绵羊胚胎的冷冻保存也获得了基本相同的效果。

The invention discloses a simple and efficient embryo vitrification freezing and thawing method, which belongs to the technical field of embryo biology. The method has the advantages of high speed, low cost, simple operation and high efficiency. The present invention uses ethylene glycol (EG) or glycerol (GL) as the main antifreeze protection agent to prepare EFS or GFS vitrification solution, the concentration is greater than 7.0Mol/L, and at room temperature (20-25°C), the embryo Pre-treat in 10-20% EG (or GL) solution for 5-10 minutes, then transfer to EFS (or GFS) solution to balance for 30s-1 minute, and then directly put into liquid nitrogen. The survival rate of mouse pronuclear embryos to hatched blastocysts cryopreserved by this method can reach about 95%, and a high implantation pregnancy rate (60-75%) has been obtained. The method also obtains basically the same effect on cryopreservation of embryos of other domestic animals such as rabbits, cattle and sheep.

简易高效胚胎玻璃化冷冻解冻方法A Simple and Efficient Embryo Vitrification and Thawing Method

技术领域technical field

本发明涉及一种胚胎冷冻解冻方法，特别是用于哺乳动物的胚胎冷冻解冻方法，属胚胎生物技术领域。The invention relates to an embryo freezing and thawing method, in particular to a mammalian embryo freezing and thawing method, which belongs to the technical field of embryo biology.

技术背景technical background

1972年Whittingham等首先利用小鼠2～8细胞胚胎冷冻保存(传统冷冻法)成功并移植后获得后代，他们采用二甲基亚砜(Dimethylsulfoxide：DMSO)为主体抗冻保护剂，浓度为1.5Mol/L，使用降温冷冻仪，以-0.3℃/min的速度降至-30℃～-35℃，然后再以-1.0℃/min的速度降至-80℃后投入液氮中。冷冻解冻后胚胎成活率为50％～70％，妊娠率为65％，产仔率为48％。该技术所使用的降温冷冻仪约4万美元/台，费用昂贵，且整个冷冻程序需3小时，操作时间长、效率低，更不适应于边远牧区的胚胎冷冻。In 1972, Whittingham et al. first used mouse 2-8 cell embryos to be cryopreserved (traditional freezing method) and obtained offspring after transplantation. They used dimethylsulfoxide (Dimethylsulfoxide: DMSO) as the main antifreeze protective agent at a concentration of 1.5Mol /L, using a cooling freezer, drop to -30°C to -35°C at a rate of -0.3°C/min, and then drop to -80°C at a rate of -1.0°C/min before putting it into liquid nitrogen. After freezing and thawing, the survival rate of embryos was 50% to 70%, the pregnancy rate was 65%, and the litter rate was 48%. The cooling equipment used in this technology is about 40,000 US dollars per unit, which is expensive, and the whole freezing process takes 3 hours, the operation time is long, the efficiency is low, and it is not suitable for embryo freezing in remote pastoral areas.

1985年Rall等又发明了胚胎玻璃化冷冻方法，他们利用DMSO，乙酰胺(Acetamide)丙二醇(Propylene glycol)为主体的抗冻保护剂，配制成浓度高达6Mol/L的玻璃化溶液(VS液)，不使用降温冷冻仪，在室温下对小鼠2细胞、8细胞和桑椹胚进行冷冻保存，该方法需要分三步降温(22℃下胚胎于25％VS液中平衡15min，4℃下于50％VS液中平衡10min，4℃下于100％VS液中平衡10min)，整个程序需要35min。该技术由于所用抗冻保护剂对胚胎的化学毒性较大，冷冻后胚胎的成活率不稳定(46％～95％)，产仔率较低(22％～38％)。In 1985, Rall et al. invented the embryo vitrification method again. They used DMSO, Acetamide (Acetamide) and Propylene glycol (Propylene glycol) as the main antifreeze protection agent to prepare a vitrification solution (VS solution) with a concentration of up to 6Mol/L. , cryopreservation of mouse 2-cell, 8-cell and morula at room temperature without using a cooling freezer, this method needs to be cooled in three steps (embryos are equilibrated in 25% VS solution at 22°C for 15min, and at 4°C in Equilibrate in 50% VS solution for 10 minutes, and equilibrate in 100% VS solution for 10 minutes at 4°C), the whole procedure takes 35 minutes. Due to the high chemical toxicity of the cryoprotectants used in this technology, the survival rate of frozen embryos is unstable (46%-95%), and the litter rate is low (22%-38%).

发明内容Contents of the invention

本发明的目的是要提供一种速度快、成本低、操作简单、效率高的胚胎冷冻解冻方法。The object of the present invention is to provide a method for freezing and thawing embryos with high speed, low cost, simple operation and high efficiency.

为了达到本发明的目的，本发明者首先对几种抗冻保护剂进行筛选，通过对胚胎的毒性试验及胚胎细胞膜的通透性测定，证明乙二醇(Ethyleneglycol：EG)的化学毒性最小且通透性强，其次是丙三醇(Glycerol：GL)、丙二醇、DMSO、乙酰胺。为此，发明者以乙二醇(或丙三醇)为主体抗冻保护剂，添加大分子物质聚蔗糖和蔗糖配制成EFS(或GFS)液，其浓度大于7.0Mol/L。为避免其对胚胎的化学毒性伤害，冷冻前将胚胎在浓度为10～20％EG(或GL)溶液中预处理5～10min，再移入EFS(或GFS)溶液中平衡30s～1min后直接投入液氮即二步法冷冻保存。目的是既能使抗冻保护剂乙二醇(或丙三醇)较为缓和地透入胚胎细胞内部，又能在冷冻的瞬间形成玻璃化状态。本方法冷冻保存的小鼠原核胚～孵化囊胚，其成活率均能达到95％左右，并获得了较高的移植妊娠率(60～75％)。本方法对其他家畜如兔、牛和绵羊胚胎的冷冻保存也获得了基本相同的效果。In order to achieve the purpose of the present invention, the inventor at first screens several cryoprotectants, and through the permeability test of embryonic cell membranes and embryonic cell membranes, it is proved that the chemical toxicity of ethylene glycol (Ethyleneglycol: EG) is minimal and Strong permeability, followed by glycerol (Glycerol: GL), propylene glycol, DMSO, acetamide. For this reason, the inventors use ethylene glycol (or glycerol) as the main antifreeze protective agent, add macromolecular substances polysucrose and sucrose to prepare EFS (or GFS) solution, and its concentration is greater than 7.0Mol/L. In order to avoid chemical toxicity damage to the embryos, the embryos are pretreated in 10-20% EG (or GL) solution for 5-10 minutes before freezing, and then transferred to EFS (or GFS) solution to balance for 30s-1min and then directly put into Liquid nitrogen is two-step cryopreservation. The purpose is not only to make the antifreeze protective agent ethylene glycol (or glycerol) penetrate into the embryonic cells more gently, but also to form a vitrified state at the moment of freezing. The survival rate of mouse pronuclear embryos to hatched blastocysts cryopreserved by the method can reach about 95%, and a relatively high implantation pregnancy rate (60-75%) is obtained. The method also obtains basically the same effect on cryopreservation of embryos of other domestic animals such as rabbits, cattle and sheep.

本发明的具体技术解决方案是：Concrete technical solution of the present invention is:

(1)配制10％～20％乙二醇(EG)溶液：取1～2ml乙二醇溶液+9～8ml PBS充分混合即成；(1) Prepare 10%-20% ethylene glycol (EG) solution: take 1-2ml ethylene glycol solution + 9-8ml PBS and mix thoroughly;

(2)配制EFS玻璃化溶液：将聚蔗糖(Ficoll 70,000)和蔗糖(Sucrose)用PBS稀释成21％～18％(FS液中聚蔗糖的含量，W/V)和0.35Mol/L～0.3Mol/L(FS液中蔗糖的含量，W/V)浓度的混合液即FS液，然后将FS液与乙二醇相应按7∶3～6∶4的比例(V/V)混合后制成EFS30～EFS40玻璃化溶液；(2) Prepare EFS vitrification solution: Dilute polysucrose (Ficoll 70,000) and sucrose (Sucrose) with PBS to 21%-18% (content of polysucrose in FS solution, W/V) and 0.35Mol/L-0.3 Mol/L (content of sucrose in FS liquid, W/V) concentration of the mixed liquid is FS liquid, and then the FS liquid and ethylene glycol are mixed according to the ratio (V/V) of 7:3 to 6:4 to prepare Form EFS30～EFS40 vitrification solution;

(3)冷冻：在室温(20～25℃)下，将胚胎移入10％～20％EG溶液中预处理5～10min，然后移入含有EFS玻璃化溶液的细管中平衡30s～1min，封口即投入液氮中冷冻保存；(3) Freezing: At room temperature (20-25°C), transfer the embryos into 10%-20% EG solution for pretreatment for 5-10 minutes, then transfer them into a thin tube containing EFS vitrification solution to balance for 30 seconds-1 minute, and seal it. put into liquid nitrogen for cryopreservation;

(4)解冻：需要解冻时，将冷冻保存的细管由液氮中取出后直接浸入20～25℃水浴中，约10s钟解冻后，用0.3～1.0Mol/L蔗糖溶液冲洗管中内容物，在显微镜下回收胚胎。(4) Thawing: When thawing is required, take out the cryopreserved thin tube from liquid nitrogen and immerse it directly in a water bath at 20-25°C. After thawing for about 10 seconds, rinse the contents in the tube with 0.3-1.0Mol/L sucrose solution , and the embryos were recovered under a microscope.

上述技术解决方案中所采用的抗冷冻保护剂乙二醇(EG)可由丙三醇(GL)代替，则其技术方案为：The anti-freeze protection agent ethylene glycol (EG) adopted in the above-mentioned technical solution can be replaced by glycerol (GL), then its technical scheme is:

(1)配制10％～20％丙三醇(GL)溶液：取1～2ml丙三醇溶液+9～8ml PBS充分混合即成；(1) Prepare 10%-20% glycerol (GL) solution: take 1-2ml glycerol solution + 9-8ml PBS and mix thoroughly;

(2)配制GFS玻璃化溶液：将聚蔗糖(Ficoll 70,000)和蔗糖(Sucrose)用PBS稀释成30％(FS液中聚蔗糖的含量，W/V)和0.5Mol/L(FS液中蔗糖的含量，W/V)浓度的混合液即FS液，然后将FS液与丙三醇(GL)按5∶5的比例(V/V)混合后制成GFS50玻璃化溶液；(2) Prepare GFS vitrification solution: Dilute polysucrose (Ficoll 70,000) and sucrose (Sucrose) with PBS to 30% (polysucrose content in FS liquid, W/V) and 0.5Mol/L (sucrose in FS liquid The mixed solution of concentration, W/V) is the FS solution, and then the FS solution and glycerol (GL) are mixed in a ratio of 5:5 (V/V) to make GFS50 vitrification solution;

(3)冷冻：在室温(20～25℃)下，将胚胎移入10％～20％GL溶液中预处理5～10min，然后移入含有GFS玻璃化溶液的细管中平衡30s～1min，封口即投入液氮中冷冻保存；(3) Freezing: At room temperature (20-25°C), transfer the embryos into 10%-20% GL solution for pretreatment for 5-10 minutes, then transfer them into a thin tube containing GFS vitrification solution to balance for 30 seconds-1 minute, and seal it. put into liquid nitrogen for cryopreservation;

(4)解冻：需要解冻时，将冷冻保存的细管由液氮中取出后直接浸入20～25℃水浴中，约10s中解冻后，用0.3～1.0Mol/L蔗糖溶液冲洗管中内容物，在显微镜下回收胚胎。(4) Thawing: When thawing is required, take out the cryopreserved thin tube from liquid nitrogen and immerse it directly in a water bath at 20-25°C. After thawing for about 10 seconds, rinse the contents in the tube with 0.3-1.0Mol/L sucrose solution , and the embryos were recovered under a microscope.

在上述的技术解决方案中，其步骤(3)的冷冻过程中所用的细管为0.25ml塑料细管。In the above-mentioned technical solution, the thin tube used in the freezing process of its step (3) is a 0.25ml plastic thin tube.

本发明具有如下优点：The present invention has the following advantages:

(1)速度快：改变传统冷冻方法的时间长、速度慢，即由一个冷冻程序3h缩短为30s；(1) Fast speed: It takes a long time and slow speed to change the traditional freezing method, that is, a freezing procedure is shortened from 3 hours to 30 seconds;

(2)降低成本、操作简单：传统方法中使用降温冷冻仪(约4万美元/台)冷冻胚胎，改为室温(20～25℃)下徒手操作；(2) Reduced cost and simple operation: In the traditional method, the embryos are frozen by using a cooling freezer (about 40,000 US dollars per set), and replaced by manual operation at room temperature (20-25°C);

(3)无冰晶：传统冷冻方法有冰晶生成，本方法无冰晶生成，即呈玻璃化状态，主要是使用了高分子物质聚蔗糖(WF：70,000)，可避免冰晶对胚胎的物理性损伤；(3) No ice crystals: The traditional freezing method produces ice crystals, but this method does not generate ice crystals, that is, it is in a vitrified state. The polymer material polysucrose (WF: 70,000) is mainly used to avoid physical damage to the embryo by ice crystals;

(4)适用范围广：传统的方法需要在实验室内进行，本方法可在现场操作，特别是在无电和边远牧区更为实用；(4) Wide range of application: the traditional method needs to be carried out in the laboratory, and this method can be operated on site, especially in remote pastoral areas without electricity;

(5)效率高：与传统的方法相比，本方法用EFS冷冻后胚胎成活率(94％～100％)、移植妊娠率(66％～75％)和产仔率(56％～66％)可提高5％～10％。用GFS冷冻后胚胎成活率为92％，移植妊娠率为60％，产仔率高达74％。(5) High efficiency: Compared with the traditional method, the embryo survival rate (94%-100%), implantation pregnancy rate (66%-75%) and litter rate (56%-66% ) can be increased by 5% to 10%. After freezing with GFS, the survival rate of embryos is 92%, the transfer pregnancy rate is 60%, and the litter rate is as high as 74%.

附图说明Description of drawings

图1为本发明的胚胎玻璃化冷冻方法的实施例示意图；Fig. 1 is the embodiment schematic diagram of embryo vitrification freezing method of the present invention;

图2为本发明的胚胎玻璃化解冻方法的实施例示意图。Fig. 2 is a schematic diagram of an embodiment of the embryo vitrification and thawing method of the present invention.

实施例Example

下面结合附图和实施例对本发明做进一步的说明。The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

如图1所示，将胚胎于PBS液中洗净，用巴氏吸管将胚胎移入10％的EG(或GL)液中，在20～25℃室温下预处理5min，然后将胚胎移入含有EFS(或GFS)液和蔗糖溶液的0.25ml塑料细管的EFS(或GFS)液段中平衡30s，用封口粉封口即投入液氮，移入液氮罐中保存。As shown in Figure 1, wash the embryos in PBS solution, transfer the embryos into 10% EG (or GL) solution with a Pasteur pipette, pretreat at room temperature at 20-25°C for 5 minutes, and then transfer the embryos into (or GFS) liquid and sucrose solution in the EFS (or GFS) liquid segment of a 0.25ml plastic thin tube for 30s, seal with sealing powder and put into liquid nitrogen, and move into a liquid nitrogen tank for storage.

如图2所示，将冷冻保存的细管由液氮中取出后直接浸入20～25℃水浴中，约10s解冻后，用0.5Mol/L蔗糖溶液冲洗管中内容物，在显微镜下回收胚胎，并将回收的胚胎移入新鲜0.5Mol/L蔗糖溶液中，再将胚胎移入PBS液中洗净后培养。以上冲洗、回收、移入步骤在5min内完成。As shown in Figure 2, take the cryopreserved thin tube out of liquid nitrogen and immerse it directly in a water bath at 20-25°C. After thawing for about 10 seconds, wash the contents in the tube with 0.5Mol/L sucrose solution, and recover the embryo under a microscope. , and transferred the recovered embryos into fresh 0.5Mol/L sucrose solution, and then transferred the embryos into PBS solution to wash and culture. The above washing, recovery, and transfer steps are completed within 5 minutes.

下面是本发明简易高效胚胎玻璃化冷冻解冻方法，的两个具体实施例：Below are two specific examples of the simple and efficient embryo vitrification and thawing method of the present invention:

例1：(1)配制10％乙二醇(EG)溶液：取1ml乙二醇溶液+9ml PBS充分混合即成；Example 1: (1) Prepare 10% ethylene glycol (EG) solution: take 1ml ethylene glycol solution + 9ml PBS and mix well;

(2)配制EFS玻璃化溶液：将聚蔗糖(Ficoll 70,000)和蔗糖(Sucrose)用PBS稀释成21％和0.35Mol/L浓度的混合液即FS液，然后将FS液与乙二醇按7∶3的比例(v/v)混合后制成EFS30玻璃化溶液；(2) Preparation of EFS vitrification solution: Dilute polysucrose (Ficoll 70,000) and sucrose (Sucrose) with PBS to a mixture of 21% and 0.35Mol/L concentration, namely FS solution, and then mix FS solution with ethylene glycol by 7 : 3 ratio (v/v) mixed to make EFS30 vitrification solution;

(3)冷冻：在室温(20～25℃)下，将胚胎移入10％EG溶液中预处理5min，然后移入含有EFS30或EFS40即玻璃化溶液的细管中平衡30s，封口即投入液氮中冷冻保存；(3) Freezing: At room temperature (20-25°C), transfer the embryos into 10% EG solution for pretreatment for 5 minutes, then transfer them into a thin tube containing EFS30 or EFS40, vitrification solution, and equilibrate for 30 seconds, seal it and put it into liquid nitrogen cryopreservation;

(4)解冻：需要解冻时，将冷冻保存的细管由液氮中取出后直接浸入20～25℃水浴中，约10s钟解冻后，用0.5Mol/L蔗糖溶液冲洗管中内容物，在显微镜下回收胚胎。(4) Thawing: When thawing is required, take out the cryopreserved thin tube from liquid nitrogen and immerse it directly in a water bath at 20-25°C. After thawing for about 10 seconds, rinse the contents in the tube with 0.5Mol/L sucrose solution, Embryos were recovered under a microscope.

例2：(1)配制10％丙三醇(GL)溶液：取1ml丙三醇溶液+9ml PBS充分混合即成；Example 2: (1) Prepare 10% glycerol (GL) solution: take 1ml glycerol solution + 9ml PBS and mix thoroughly;

(2)配制GFS玻璃化溶液：将聚蔗糖(Ficoll 70,000)和蔗糖(Sucrose)用PBS稀释成30％和0.5Mol/L浓度的混合液即FS液，然后将FS液与丙三醇(GL)按5∶5的比例(V/V)混合后制成GFS50玻璃化溶液；(2) Preparation of GFS vitrification solution: Dilute polysucrose (Ficoll 70,000) and sucrose (Sucrose) with PBS to a mixture of 30% and 0.5Mol/L concentration, namely FS solution, and then mix FS solution with glycerol (GL ) were mixed at a ratio of 5:5 (V/V) to make GFS50 vitrification solution;

(3)冷冻：在室温(20～25℃)下，将胚胎移入10％GL溶液中预处理5min，然后移入含有GFS50玻璃化溶液的细管中平衡30s～1min，封口即投入液氮中冷冻保存；(3) Freezing: At room temperature (20-25°C), transfer the embryos into a 10% GL solution for pretreatment for 5 minutes, then transfer them into a thin tube containing GFS50 vitrification solution to balance for 30 seconds-1 minute, seal and freeze in liquid nitrogen save;

(4)解冻：需要解冻时，将冷冻保存的细管由液氮中取出后直接浸入20～25℃水浴中，约10s解冻后，用0.5Mol/L蔗糖溶液冲洗管中内容物，在显微镜下回收胚胎。(4) Thawing: When thawing is required, take out the cryopreserved thin tube from liquid nitrogen and immerse it directly in a water bath at 20-25°C. After thawing for about 10 seconds, rinse the contents in the tube with 0.5Mol/L sucrose solution and place it under the microscope. Embryos were recovered.